Biomass can be a viable supplement and alternative to non-renewable sources of fuel and chemicals. Lignin is an important part of biomass sources which can be used in various chemical and fuel industries. This study explores the pretreatment of lignin from Napier grass using thermal and physical means, as well as extraction of lignin via cellulolytic enzymatic hydrolysis to determine the optimum condition for feedstock pretreatment. Napier grass parts under various drying conditions and particle sizes were treated with enzymes. Moisture analysis, FTIR spectroscopy, UV−Vis analysis, and Klason lignin were carried out to analyze the moisture, functional group, and yield of lignin. Moisture content of the samples were inversely proportional to the drying conditions. The FTIR result showed lower peak intensity for higher drying conditions, while ball-milling showed less reduction in peak intensity. More Klason lignin was extracted under higher drying conditions. The yield of cellulolytic enzymatic lignin (CEL) was found to be more than actual lignin content, suggesting cellulose was not fully degraded. The FTIR spectra of CEL was found to be closer to that of lignin, but purification was still needed. Optimization was carried out by evaluating the statistical significance of each pretreatment effect of the pretreatments.